Proteins from plants, animals and common bacteria have been extensively studied in recent years, and structural work has revealed many common motifs in these proteins. Less common organisms, especially those which flourish under extreme environmental conditions (for example, thermophiles and acidophiles) might be expected to have unusual structural motifs in their proteins. One chemolithotrophic organism which has received a great deal of attention recently is Thiobacillus ferrooxidans, which oxidizes ferrous ions under extremely acid conditions (pH ~1.5). The organism utilizes in its electron transfer pathway a copper protein, rusticyanin, which is produced at high levels and has been characterized as a "blue" copper protein with several unusual features, including atypical EPR spectrum and reduction potential and pH optimum ~1.5. The amino acid sequence has recently been published, but no three-dimensional structure is available. It has a MW of 16,500 and preliminary NMR spectra reveal that it is an ideal candidate for structure determination. The protein can be readily and relatively cheaply labeled with stable isotopes in the T. ferrooxidans system, using NaH13CO3 and (15NH4)2SO4. Preliminary 1H NMR spectra of unlabeled rusticyanin reveal it to be monomeric at pH 1.5, with narrow linewidths and favorable dispersion of resonances. We propose to undertake a structural analysis of this highly unusual protein, using NMR spectroscopy in solution at the acidic pH optimum. Given the quality of the NMR spectra and the availability of labeled material, we are confident that a high-resolution structure will be obtained, from which we can deduce the nature of the copper coordination sphere. Relaxation measurements will be undertaken to explore the internal dynamics of the molecule in solution. Comparisons with other Type I copper proteins will be made to elucidate the factors which promote the stability of rusticyanin at acidic pH, as well as the structural basis of the unusual electronic properties of rusticyanin. As well as being of intrinsic interest as a member of the family of copper proteins, we anticipate that structural information on rusticyanin and other proteins from thermophilic and acidophilic organisms will increase our understanding of the factors responsible for the unusual stability and electron transfer properties at low pH. Such knowledge could be applied to protein design for medical and industrial use.